The utility of full-length transcripts for cancer research

The facility of nanopore sequencing to deliver full-length transcripts and to accurately identify and quantify multiple isoforms has made the technology particularly useful for cancer research. The technology has been applied to the study of many cancer types including, leukaemia¹, breast cancer², ovarian cancer², and lung cancer³.

Suzuki et al.³ employed nanopore cDNA sequencing to detect a variety of common mutation types, including single base substitutions, short deletions, exon skipping, and fusions, in a set of genes implicated in lung cancer (EGFR, KRAS, NRAS, NF1, ALK and RET). Further, the team utilised the long-reads delivered by nanopore sequencing to characterise the phasing of two genomic DNA mutations that are associated with anti-cancer drug efficacy. Following this successful study, the researchers commented that: ‘The simple methods of MinION sequencing could possibly enable small/mid-scale research centers and hospitals to conduct research studies by genotyping driver genes and selecting suitable therapeutic approaches’ ³.

At the University of Bari, Italy, researchers are investigating the potential of nanopore sequencing to detect BCR-ABL1 kinase domain (KD) mutations¹. The BCR-ABL1 gene fusion, which codes for a non-regulated tyrosine kinase protein, occurs in 95% of people with chronic myeloid leukaemia (CML)⁴. KD mutations in the BCR-ABL1 gene contribute to and indicate resistance to first-line therapy with tyrosine kinase inhibitors¹. The current gold-standard test for variant detection in BCR-ABL1 is Sanger sequencing; however, with a sensitivity of just 20%, it is unsuitable for identifying low-level variants.

‘…MinION is markedly superior to Sanger sequencing in terms of sensitivity, costs and timesaving…’¹

In a 24-sample study, the team assessed the performance of nanopore sequencing to identify KD mutations in a 1.7 kb cDNA amplicon at both 100x and 1,000x sequencing depth. The nanopore data allowed the identification of clinically important mutations in 2 samples which were not initially evident using Sanger sequencing (Figure 1). The team also reported that the lower nanopore sequencing depth of 100x was sufficient to identify all mutations. The long reads delivered by nanopore sequencing further enabled the detection of mutations that are in the same clone (in cis; compound mutants), something which is particularly challenging for Sanger or short-read sequencing approaches. The researchers summarised that: ‘Our data indicates that MinION is markedly superior to Sanger sequencing in terms of sensitivity, costs and timesaving, and has the added advantage of determining the clonal configuration of multiple mutations’ ¹.